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Optimizing accuracy and depth of protein quantification in experiments using isobaric carriers

View ORCID ProfileHarrison Specht, View ORCID ProfileNikolai Slavov
doi: https://doi.org/10.1101/2020.08.24.264994
Harrison Specht
1Department of Bioengineering, Northeastern University, Boston, MA 02115, USA
2Barnett Institute, Northeastern University, Boston, MA 02115, USA
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  • ORCID record for Harrison Specht
Nikolai Slavov
1Department of Bioengineering, Northeastern University, Boston, MA 02115, USA
2Barnett Institute, Northeastern University, Boston, MA 02115, USA
3Department of Biology, Northeastern University, Boston, MA 02115, USA
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  • ORCID record for Nikolai Slavov
  • For correspondence: nslavov@alum.mit.edu nslavov@northeastern.edu
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Abstract

The isobaric carrier approach, which combines small isobarically-labeled samples with a larger isobarically-labeled carrier sample, is finding diverse applications in ultrasensitive mass-spectrometry analysis of very small samples, such as single cells. To enhance the growing use of isobaric carriers, we characterized the trade-offs of using isobaric carriers in controlled experiments with complex human proteomes. The data indicate that isobaric carriers directly enhances peptide sequence identification without simultaneously increasing the number of protein copies sampled from small samples. The results also indicate strategies for optimizing the amount of isobaric carrier and analytical parameters, such as ion accumulation time, for different priorities such as improved quantification or increased number of identified proteins. Balancing these trade-offs enables adapting isobaric carrier experiments to different applications, such as quantifying proteins from limited biopsies or organoids, building single-cell atlases, or modeling protein networks in single cells. In all cases, the reliability of protein quantification should be estimated and incorporated in all subsequent analysis. We expect that these guidelines will aid in explicit incorporation of the characterized trade-offs in experimental designs and transparent error propagation in data analysis.

Figure

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • ∈ Data & analysis code: scope2.slavovlab.net

  • We added an additional figures and analysis reporting on the number of peptide fragments per peptide. We also added a new figure 8 showing the effects of directly decreasing or increasing ion accumulation times on missing data and quantification accuracy. There is no change in the principal findings and guidelines.

  • http://scope2.slavovlab.net/

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted October 21, 2020.
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Optimizing accuracy and depth of protein quantification in experiments using isobaric carriers
Harrison Specht, Nikolai Slavov
bioRxiv 2020.08.24.264994; doi: https://doi.org/10.1101/2020.08.24.264994
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Optimizing accuracy and depth of protein quantification in experiments using isobaric carriers
Harrison Specht, Nikolai Slavov
bioRxiv 2020.08.24.264994; doi: https://doi.org/10.1101/2020.08.24.264994

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